Vascular inflammation and atherosclerosis are implicated in many cardiovascular diseases. While the systemic risk factors such as hyperlipidemia are exposed to entire vasculature under pathological conditions, the atherosclerotic plaques often preferentially develop at sites with disturbed flow, indicating a role of hemodynamic forces in atherogenesis. For decades, endothelial cell (EC) responses to the changes in wall shear stress (WSS) have been the main, if not sole, focus of the flow dynamics related studies, which basically consider the circulating blood as a cell-free fluid and completely overlooked the impact of mechanical force-activated blood cells on the vascular walls. Our recent study conducted in intact venules showed that changes in blood flow alter EC function through both WSS and the SS-induced release of ATP from RBCs, and the RBC released ATP through a pannexin 1 (Panx1) channel plays a key role in altering EC barrier integrity. This novel observation led us to hypothesize that the RBC-released ATP during blood flow changes plays a significant role in site-specific vascular vulnerability and synergistically contributes to the initiation and progression of vascular inflammation and atherosclerosis along with local WSS and systemic risk factors. This hypothesis will be tested experimentally in vivo and computationally in silico under three specific aims using two newly established hypercholesterolemia mouse models with blood cell specific deletion of Panx1 (ApoE-/-Panx1-/- and AAV- PCSK9DYPanx1-/- fed with high fat diet). Aim 1 is to investigate the role of RBC-released ATP in the site- specific endothelium vulnerability to inflammation; Aim 2 is to investigate the contribution of RBC released ATP to hypercholesterolemia-induced site-specific plaque formation and atherosclerosis progression in major arteries. Our preliminary data showed about 40-60% reduction of aorta atherosclerotic plaque at branch regions in mouse with RBC Panx1 deletion, suggesting an important role of RBC released ATP in disturbed blood flow-initiated vascular pathogenesis. The potential roles of RBC released ATP in blood immune cell alterations, plasma microparticles and cytokine levels will also be investigated. Aim 3 is to utilize a high-fidelity, three-dimensional, multiscale computational model to predict the distribution of SS on the RBC membrane, the stress-induced ATP release, and the distribution of ATP at the vascular walls. Complementing the in vivo studies proposed in Aims 1 and 2, the proposed computational studies will provide, for the first time, a distinction between the roles of RBC stress and WSS in both micro and macro- circulation under diverse flow conditions, and hence, hemodynamic insights into RBC-mediated vascular pathogenesis. The proposed study challenges the conventional views in the field and will provide a more comprehensive characterization of local hemodynamic and systemic environmental factors responsible for vascular pathogenesis and aid the development of novel therapeutics.